Alobophora sandrae n. gen. n. sp. (Digenea: Caballerotrematidae) infecting Arapaima gigas sensu lato (Osteoglossiformes: Arapaimidae) with a revision of Caballerotrema, key to Caballerotrematidae, and updated phylogeny

We propose and describe Alobophora sandrae Cajiao-Mora & Bullard n. gen., n. sp. (Digenea: Caballerotrematidae) for specimens we collected from arapaima, Arapaima gigas sensu lato (Osteoglossiformes: Arapaimidae) in the Amazon River near Leticia, Colombia. Alobophora differs from Caballerotrema Prudhoe, 1960 by lacking head collar projections and by having clustered corner spines and a narrow head collar (4–5× wider than pharynx), whereas Caballerotrema has head collar projections, lacks clustered corner spines, and has a broad head collar (7–8× wider than pharynx). We reassign Caballerotrema annulatum (Diesing, 1850) Ostrowski de Núñez & Sattmann, 2002 to the new genus, as Alobophora annulata (Diesing, 1850) Cajiao-Mora and Bullard n. comb., and provide a supplemental description of Caballerotrema brasiliense Prudhoe, 1960 based on specimens we collected from arapaima. We also examined the holotype and a paratype of Caballerotrema piscicola (Stunkard, 1960) Kostadinova & Gibson, 2001 and concluded that C. piscicola is a junior subjective synonym of C. brasiliense. Our 28S phylogeny recovered A. sandrae sister to A. annulata, with that clade sister to a clade comprising C. brasiliense and an innominate species of Caballerotrema. Caballerotrematidae was recovered sister to Echinostomatidae. We also provide a dichotomous key to caballerotrematids based on head collar projections, corner spine arrangement, proportional pharynx and head collar breadth, testes shape and arrangement, body surface spine shape and distribution, vitellarium distribution, and abundance of prostatic cells.

The host we report herein, "Arapaima gigas" sensu lato (Arapaimidae), is among the largest freshwater fishes in the world, reaching 3 m in total length and 250 kg [73].Arapaimas are piscivorous, facultative air-breathing fish with a swim bladder that functions as a breathing organ [8].They naturally range in the central Amazon region: the floodplain of the rivers Araguaia-Tocantins, Solimões-Amazonas in Brazil, Colombia, Peru, and in the Essequibo and Rupununi river system of Guiana [13,14,38].Their theorized invasive distribution (human-mediated) comprises rivers in Peru, Brazil, Bolivia, India, and Indonesia [15,28,47,58,61,71].These iconic fish are culturally and commercially important [36].Their abundances are alleged to be declining as indicated by decreased landings at artisanal fish markets [36,38].Nevertheless, arapaima has been catalogued as "Data Deficient" since 1996 in the Red List of Threatened Species of the International Union for Conservation of Nature (IUCN) [93].The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) [89] lists it as Appendix II ("Not necessarily now threatened with extinction but could become so unless trade is closely controlled").Some authors have classified arapaima as a "conservation paradox" because of its "threatened" status in its native range (Brazil) and its invasive status in its theorized invasive range [15,58].
The current taxonomic status of "Arapaima gigas" is contentious.Günther [34] synonymized the three species of Arapaima described by Valenciennes [90] i.e., Arapaima agassizii Valenciennes and Arapaima mapae Valenciennes from Brazil plus Arapaima arapaima Valenciennes from the Rupununi River in Guyana.Stewart [81] rejected the synonymies of Günther [34] and described a fifth species (Arapaima leptosoma Stewart) from the Solimões River, Amazonas, Brazil, based upon a combination of features associated with the morphology of the preopercle and dorsal fin.Although Fricke et al. [30] accepted the 5 species of Arapaima, the vast majority of modern, non-taxonomic literature pertaining to arapaimas still references the single species "A.gigas" in the broad sense (sensu lato).Genetic studies of arapaimas in the ARB indicated the presence of metapopulations, not distinct arapaima species [3,29,37,63,87,91].This evolving understanding of arapaima evolution and population biology makes the study of their parasites intriguing since parasites can be indicators of cryptic host species.
Herein, we provide a description of a new species of Caballerotrematidae, propose a new genus for the new species, and propose a new synonymy for a closely related species.We also provide and updated phylogenetic analysis with the first sequences of the type species of Caballerotrema.

Ethics
All applicable institutional, national, and international guidelines for the care and use of animals were followed.Fishes were acquired post-mortem from the local market.

Parasite collection
Two fresh-dead, iced arapaima and the intestine of another fresh-dead, iced arapaima were collected opportunely from Plaza de Mercado (4°12 0 56.29 00 S 69°56 0 40.15 00 W), Leticia, Amazonas, Colombia in October 2023.Each intestine was dissected such that the intestinal mucosa was exposed before being placed in an acrylic settling column, exposed to tap water heated to 60 °C, and then rinsed by grasping the intestine with hemostats and repeatedly and rapidly dunking the intestine in the hot water.The intestine was then examined in a dish of clean citrated saline solution using a stereo-dissecting microscope with fiber optic lights and sub-stage illumination.Simultaneously, the washed contents of the intestine were allowed to settle in the acrylic column.After ~10 min, the flocculant material and fluid in the acrylic column was decanted and fresh water added.The sediment of the acrylic column then was pipetted incrementally into a clean glass petri dish with tap water and examined using a stereo-dissecting microscope.Heat-killed trematode specimens from the petri dish were then transferred with an artist brush or pipette into 10% neutral buffered formalin (n.b.f.) for morphology or into 95% non-denatured ethanol (EtOH) for DNA extraction.Fixed specimens were rinsed in water then stained overnight in Van Cleave's hematoxylin with several drops of Ehrlich's hematoxylin, dehydrated with a graded EtOH series, made basic at 70% EtOH with lithium carbonate and n-butylamine, dehydrated in absolute EtOH, cleared with clove oil, and permanently mounted on glass slides using Canada balsam and a coverslip.These specimens were drawn using an Olympus BX51 compound microscope (Olympus, Tokyo, Japan) equipped with differential interference contrast optical components and a drawing tube.Measurements were obtained using a Jenotipik Gryphax camera (Jenotipik AG, Jena, Germany) and are reported in micrometers (lm; unless otherwise stated) as the range followed by the mean, standard deviation, and sample size in parenthesis (Tables 1 and 2).Only spines in near optimal or optimal lateral or dorso-ventral view were measured (Table 2).Morphological terms and nomenclature for the genus follow Prudhoe [68], Ostrowski de Núñez and Sattmann [66], and Kostadinova and Gibson [44], except for the terminology of the esophagus that follows Truong et al. [88].Terminology for the head collar follows Kanev et al. [40] and Cajiao-Mora et al. [11], and shape names follow Clopton [18].Type specimens (1 holotype and 2 paratypes) of the new species and 4 vouchers of C. brasiliense were deposited in the National Museum of Natural History's Invertebrate Zoology Collection (USNM, Smithsonian Institution, Washington, DC).An additional paratype of the new species and 2 vouchers of C. brasiliense were deposited in the Animal Parasitological Collection (APC, Agrarian Science Department, Universidad de Antioquia, Medellín, Antioquia, Colombia).

DNA extraction
A total of 4 EtOH-preserved specimens were used for DNA extraction and sequencing.Extraction was made using a DNea-syTM Blood and Tissue kit (QIAGEN, Hilden, Germany) following the manufacturer's protocol, except that the proteinase-K incubation period was extended overnight.Once extracted, DNA concentration was measured using a NanoDrop-One Microvolume Spectrophotometer (Thermo Fisher Scientific Waltham, MA, USA), diluted to 50 ng/lL, and stored at À20 °C.The partial 28S and ITS2 were amplified using primers outlined in Anderson et al., Cribb et al., Lockyer et al., all in Cajiao-Mora et al. [1,11,20,52].PCR reactions were performed following Truong et al. [88].DNA amplification was verified with a 1% agarose gel stained with ethidium bromide.PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer's protocol, except that the last elution step was performed with autoclaved nanopure H 2 O.DNA sequencing was performed by Genewiz (South Plainfield, NJ, USA).Sequence assembly and analysis of chromatograms were performed with Geneious prime version 2023.2.1.Nucleotide sequences of the new species were 1526 base pairs (bp) for the 28S and 404 bp for the ITS2; those of C. brasiliense were 1520 bp for the 28S and 483 bp for the ITS2.The obtained ITS2 sequences were not used in the present study because there are insufficient available data in GenBank to make comparisons.All sequence data were deposited in GenBank.

Remarks
Alobophora differs from Caballerotrema by having a narrow head collar (4-5Â wider than pharynx) and clustered corner spines and by lacking head collar projections, whereas Caballerotrema has a broad head collar (7-8Â wider than pharynx), lacks clustered corner spines, and has head collar projections.We herein reassign C. annulatum to the new genus as Alobophora annulata (Diesing, 1850) Cajiao-Mora and Bullard n. comb.because it lacks head collar projections and because of the arrangement of its corner spines.Hence, we accept 2 species of Alobophora: the type species (A.sandrae) and A. annulata.The new species differs from its congener (A.annulata) by the combination of having a head collar that is wider than the maximum body width, body surface spines that decrease in size posteriad and terminate in the anterior body half (anterior to testes), a cirrus sac having few prostatic cells, and a vitellarium that extends anteriad to the cirrus sac.Alobophora annulata has a body that is wider than the head collar (body widest at level of testes), body surface spines that increase in size posteriad and terminate in the posterior body half (posterior to testes), a cirrus sac having many prostatic cells, and a vitellarium that is far posterior to the cirrus sac.
Although the new species is the fourth caballerotrematid described from arapaima, the previous 3 caballerotrematids have taxonomic problems.We accept A. sandrae and C. brasiliense but we regard C. piscicola as a junior subjective synonym of C. brasiliense and follow Kostadinova and Gibson [44] in considering C. arapaimense as a species inquirendum.The justifications for these nomenclatural actions are as follows.First, we consider C. piscicola a junior subjective synonym of C. brasiliense because it has head collar projections, a wide head collar (wider than maximum body width; 7Â wider than pharynx), corner spines arranged as two separated pairs (Fig. 3F), sinuous and overlapping testes (Fig. 3D), and a vitellarium extending anteriad midway between the cirrus sac and the ovary (Fig. 3D).Prudhoe [68] described C. brasiliense infecting an arapaima from an unspecified location in the Brazilian ARB.That same year, Stunkard [83] described C. piscicola (as H. piscicola) from an arapaima also from an unspecified location in the Brazilian ARB.Stunkard's [83] specimens were placed in water for some days before they were processed for whole-mounting.As Stunkard [83] indicated, we agree that these specimens likely partly deteriorated in water because the body shape is greatly extended and the body surface spines are missing (perhaps the spines detached as the specimens deteriorated in water) [83; p. 546].He described the head collar as reniform-shaped but did not describe projections nor do the drawings show that feature (Fig. 15 in Stunkard [83]).Kostadinova and Gibson [44] reassigned H. piscicola to Caballerotrema but did not provide a morphological description of that species.We clearly discerned head collar projections in Stunkard's [83] types (USNM 1339898) of H. piscicola, (Fig. 3D-3F); however, no additional detail of the female genitalia, esophagus, esophageal bifurcation, genital pore, and body surface could be discerned from these specimens (Fig. 3D).Second, the description of C. brasiliense by Prudhoe [68] was made with poorly fixed (contracted) specimens.We herein collected new specimens, fixed them properly, and were able to provide a detailed description of the head collar, head collar spines, genitalia (cirrus sac, seminal vesicle, oviduct, Laurer's canal, oötype, vitellarium, and transverse vitelline duct), and body surface spines (distribution, measurements, and shape).This is the first diagnosed C. brasiliense tethered to a nucleotide sequence deposited in GenBank with voucher specimens in a lending museum.Third, regarding C. arapaimense, Thatcher [85] described this species from an arapaima from the Lago Janauacá, Manaus, Amazonas, Brazil.He differentiated the species from its congeners by having larger head collar corner spines.Based on the published description of C. arapaimense, Kostadinova and Gibson [44] stated that C. arapaimense could be a synonym of C. brasiliense because "it agrees well with the description of the latter" [44; p. 198].Further, Kostadinova and Gibson [44] identified one of the paratypes of C. arapaimense (INPA 038 g) as C. brasiliense based on the size and shape of its collar spines.They also mentioned that the additional paratypes (INPA 038 e; INPA 038 h) probably belong to different Caballerotrema spp.based on the different sizes and shapes of their head collar spines [44; p. 198].They considered C. arapaimense as a species inquirendum [44; p. 203], and we follow that herein.

Phylogenetic results
Our amplified 28S sequence representing A. sandrae comprised 1529 nucleotides (GenBank accession No. PQ114582) and was 97.1% similar (44 bp different) to that of A. annulata (GenBank accession No. PP256047; from Electrophorus cf.varii from the ARB in Leticia, Amazonas, Colombia).Our 28S sequences of C. brasiliense (GenBank accession No. PQ114584; PQ114585) comprised 1520 nucleotides and are identical to each other.They were 99.8% similar (3 bp different) to that of Caballerotrema sp.(GenBank Accession No. KT956941; from A. gigas from the Peruvian Amazon).The BI analysis (Fig. 4) recovered our sequence of A. sandrae sister to that of A. annulata (GenBank accession No. PP256047).They both were recovered sister to a clade comprising our sequences of C. brasiliense and that of Caballerotrema sp.(GenBank accession No. KT956941).Both clades comprise Caballerotrematidae, which was recovered sister to Echinostomatidae (see Fig. 4).Our tree topology (Fig. 4) resembles that recovered by Tkach et al. [86].It differs by having a better support value for the clade of Caballerotrematidae and Echinostomatidae (BI support values 0.99 vs 0.72).Both were recovered sister to Fasciolidae.Our tree topology differs from that of Cajiao-Mora et al. [11], who recovered Caballerotrematidae sister to Fasciolidae and both sister to Echinostomatidae, however, with low support (0.77).

Discussion
The biogeography of osteoglossiform fishes makes the study of their parasites interesting.Some authors have proposed that particular parasites of osteoglossiforms were "Gondwana relicts" [55,56], e.g., the cestode genus Nesolecithus Poche, 1922 (Amphilinidea) [32,67] and the nematode genus Nilonema Khalil, 1960 (Philometridae) [42,76].However, recent studies have challenged the hypothesis of the breakup of Gondwana as the origin of the distribution pattern in Osteoglossiformes and several other classically called K. Cajiao-Mora et al.: Parasite 2024, 31, 55 "Gondwanaland" taxa [22,49,74].A fossil-based estimate of origin time for Osteoglossomorpha ranges from the Late Triassic to the Middle Jurassic [12].The clade is old enough to have been affected by the breakup of Gondwana, and even Pangea.Nevertheless, Capobianco and Friedman [12] stressed that osteoglossomorphs are characterized by a complex biogeographic history that involved several long-distance dispersals as well as continental vicariance and that has been partially hidden by regional extinctions [12; p. 683].These intriguing aspects of osteoglossiform natural history could be explored independently by studying their parasites if the biodiversity of their parasites was better known.For example, analogous studies have used parasite taxonomic and phylogenetic evidence to test the marine incursion hypothesis of turtles in South America [10]; others explore patterns and processes of historical biogeography by combining phylogenetics and biogeography of fishes (sturgeons) and their parasites with the geological history of the Earth [17].
The identification of the arapaimas we dissected was indeterminate and nuanced based on recent taxonomic and genetic work with arapaimas.Studies on population genetics of arapaima conducted in the main stem of the Amazon River (Peru, Colombia, Brazil) and the Araguaia-Tocantins River basin (Brazil) concluded that genetic data do not support the five described species [3,29,37,63,87,91].Instead, they suggested that arapaimas show structured populations with low gene flow, a high level of relatedness, inbreeding, and reduced genetic variability [3,15,29,37,63,87,91].The results have been linked with the potential occurrence of population bottlenecks associated with genetic drift, historical reductions in stocks, the sedentary behavior of the species, and the characteristics of each basin and its floodplain dynamics [3,63,91].The pattern of genetic structure of arapaima has also been associated with the evolution of the landscape of the Amazon region [63].This is supported for arapaima populations inhabiting the lowland intercatonic basin and the Brazilian shield of the ARB.However, a study conducted in southwestern Guyana (Essequibo and Branco River basins in the Guyana shield) indicated allopatric differentiation, suggesting sympatric species inhabiting the Essequibo and Pirara Rivers [92].Hence, while the parasites of these fishes are important and could advance our understanding of arapaima natural history, we strongly suggest that a genetic voucher, photographs, and meristics are curated along with the parasite voucher or type specimens.
Infections of Caballerotrema spp.are known from cultured arapaima [4,23,79,84].Delgado et al. [23] reported C. arapaimense from the stomach of juvenile and adult arapaimas in Peru.Those records are unaccompanied by a voucher specimen or a morphological diagnosis for the parasite reported.Further, C. arapaimense has been considered a species inquirendum since Kostadinova and Gibson [44].Hence, the identification of C. arapaimense infecting cultured arapaimas in Peru remains dubious.Serrano-Martínez et al. [79] and Tafur and Cotrina [84] reported C. brasiliense and Caballerotrema sp., respectively, infecting the intestine of juvenile arapaimas cultured in the Peruvian Amazon.Fewer reports exist in wild arapaimas.Dos Santos et al. [27] identified C. brasiliense infecting the intestine of arapaimas from the Araguaia River, Mato Grosso, Brazil.Voucher specimens are curated at the Instituto Oswaldo Cruz Helminthological Collection [27].Additional records include Prudhoe [68], while erecting Caballerotrema from an arapaima from an unknown locality in Brazil, and Thatcher [85], who described C. brasiliense and C. arapaimense from arapaimas from Lago Janauacá, Manaus, Amazonas, Brazil.Comprehensive and detailed descriptions, nucleotide sequences, and voucher material curated in a museum are important for taxonomy.Prior to this study, no comprehensive morphological and nucleotide analysis had been conducted on arapaima caballerotrematids.The present description of A. sandrae and the identification of C. brasiliense represent the first records of caballerotrematids infecting arapaimas in the Colombian Amazon River.

Conclusion
We proposed and described a new genus and species of Caballerotrematidae infecting A. gigas sensu lato from the Amazon River, Colombia.We accepted two caballerotrematid species infecting arapaimas (C.brasiliense and A. sandrae), synonymized C. piscicola with C. brasiliense, reassigned C. annulatum to Alobophora, and provided a dichotomous key to Caballerotrematidae.The biodiversity of trematodes infecting bony tongues and their relatives (Osteoglossiformes) is undersampled.The parasites of these fishes have the potential to aid in testing hypotheses regarding host biogeography and natural history.

Figure 4 .
Figure 4. Large subunit ribosomal (28S) DNA phylogeny (Bayesian inference).Values beside nodes are posterior probability.Scale bar is in substitutions per site.GenBank accession numbers are in parenthesis following each taxon.Newly generated sequences of Caballerotrematidae (Digenea: Echinostomatoidea) are highlighted in bold.The new described genus and species is indicated by an arrow.Type species are indicated by asterisk (*).